Quality assessment on Polygoni Multiflori Caulis using HPLC/UV/MS combined with principle component analysis.

Zhao et al. Chemistry Central Journal 201 3, 7:1 06
http://journal.chemistrycentral.eom/content/7/1 /1 06

f^f* Chemistry Central

Journal

RESEARCH ARTICLE Open Access

Quality assessment on Polygoni Multiflori Caulis
using HPLC/UV/MS combined with principle
component analysis

Yang Zhao 1 , Chun-Pin Kao 2 , Yuan-Shiun Chang 1,3 " and Yu-Ling Ho 4 "

Abstract

Background: Polygoni Multiflori Caulis, the dried caulis of Polygonum multiflorum Thunb., is one of the commonly
used traditional Chinese medicines having antioxidant, anti-obesity, anti-inflammatory and antibacterial effects.
Polygoni Multiflori Caulis used clinically or circulated on market have great differences in their diameters. However,
to the best of our knowledge, no study has been reported on the qualities of Polygoni Multiflori Caulis with
different diameters.

Results: Systematic HPLC/UV/MS chromatographic fingerprinting and quantitative analytical methods combined
with principal component analysis were developed and applied to analyze different Polygoni Multiflori Caulis
samples. The contents of 2,3,5,4'-tetrahydroxystilbene-2-0-/3-D-glucoside, the chemical marker for quality control on
Polygoni Multiflori Caulis specified in Chinese Pharmacopoeia (2010 edition), were found to have surprising
relevance with the samples' diameters for the first time.

Conclusion: The finding provides a scientific basis for collecting Polygoni Multiflori Caulis in the best time.
Moreover, the diameter can be used as the criterion for quality control on Polygoni Multiflori Caulis as a preliminary
step in the future. In addition, scores plot obtained from principal component analysis shows the obvious
differences between unqualified Polygoni Multiflori Caulis samples and qualified ones visually, which can be used to
single out the unqualified ones with qualified ones efficiently and immediately.

Keywords: Polygoni Multiflori Caulis, Quality assessment, HPLC/UV/MS, Principle component analysis

Background

Polygoni Multiflori Caulis (PMC), Shou-Wu-Teng in
Chinese, is the dried caulis of Polygonum multiflorum
Thunb. It is one of the commonly used traditional Chinese
medicines (TCMs) listed in Chinese Pharmacopoeia (CP)
(2010 edition) [1]. Pharmacological studies indicated that it
had antioxidant [2,3], anti-obesity [4], anti-inflammatory
and antibacterial effects [5].

Anthraquinones, flavonoids and stilbene glycosides are
considered to be the main active constituents in PMC [6,7].
However, unlike Polygoni Multiflori Radix, He-Shou-Wu in

* Correspondence: yschang@mail.cmu.edu.tw; elaine@sunrise.hk.edu.tw
department of Chinese Pharmaceutical Sciences and Chinese Medicine
Resources, College of Pharmacy, China Medical University, Taichung 40402,
Taiwan

department of Nursing, Hungkuang University, Taichung 43302, Taiwan
Full list of author information is available at the end of the article

Chinese, there are only a few reports on quality control of
PMC. High performance liquid chromatography (HPLC)
with ultraviolet detector (UV) were applied to determine
the contents of 2,3,5,4 '-tetrahydroxystilbene-2-0-/?-D-
glucoside (THSG) and emodin [8-10], but THSG, one
of the stilbene glycoside, was not specified as chemical
marker for quantitative determination of PMC until CP
(2010 edition) was published.

As for original plant morphology, it is regulated in CP
(2010 edition) that the diameter of PMC is between 4
and 7 mm. However, it derives from P. multiflorum which
is a perennial plant and can be harvested all the year
round, so PMC circulated on market have big variations
on their diameters. In the PMC samples we collected, the
smallest diameter is just 0.5 mm, however, the biggest one
reaches to 36 mm. Their differences go so far to 70 times
unexpectedly. In that way, are there any differences on
their qualities? The issue arouses our great interest.

(a

© 2013 Zhao et al.; licensee Chemistry Central Ltd. This is an Open Access article distributed under the terms of the Creative
Chemistry Central Commons Attribution License (http://creativecommons.Org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

Zhao et al. Chemistry Central Journal 201 3, 7:1 06
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Page 2 of 13

In the present study, chromatographic fingerprinting
and quantitative analytical methods were developed to
analyze different PMC samples. Seven peaks, marked
as 1 to 7, were designated as characteristic peaks in
chromatographic fingerprints. They were identified as
THSG, emodin-8-0-/?-D-glucoside, emodin-8-0-(6'-
0-malonyl)-/?-D-glucoside, physcion- 8 -0-/?-D-glucoside,
physcion-8-0-(6'-0-acetyl)-/?-D-glucoside, emodin and
physcion, respectively, based on UV and MS data com-
pared with reference compounds and related literatures
[11-17]. THSG, emodin and physcion were quantified at
their maximal UV wavelengths. From the results, we
found that the contents of THSG had great relevance with
the diameters of PMC samples. Principal component
analysis (PCA), one of the popular chemometrics, was
then used for comprehensive and systematic assessment
on PMC samples collected from different regions with
different diameters, based on the variables including the
contents of the three quantified analytes and the PA/W
(peak area divided by sample weight) values of the four
unquantified ones. Very useful information were obtained
from PCA scores plot, by which unqualified PMC samples
could be distinguished from qualified ones visually and
immediately. Points of view how variables contributed to
samples' positions in scores plot were also discussed in
detail according to PCA loadings plot.

Experimental

Chemicals, solvents and herbal materials

THSG, emodin and physcion were purchased from
Shanghai R&D Center for Standardization of Traditional
Chinese Medicines. LC-grade methanol, acetonitrile, formic
acid and phosphoric acid were purchased from the branch
company of Merck in Taipei, Taiwan. Purified water was
prepared with Milli-Q system (Millipore, Milford, MA,
USA). All other reagents used in the present study were of
analytical grade. Herbal materials of PMC were collected
from different regions of mainland China and local
pharmacies of Hong Kong, which were marked as PMC-01
to PMC-08 and L-PMC-01 to L-PMC-11, respectively. The
detailed information of the samples is summarized in
Table 1. All the plant specimens have been deposited in
Department of Chinese Pharmaceutical Sciences and
Chinese Medicine Resources, School of Pharmacy,
China Medical University.

Sample preparation

Dried PMC samples were sliced into small pieces and
were ground into fine powders (20 mesh) using a grinder
with a knife blade. Half gram of each PMC powder was
carefully weighed into a 50 mL centrifuge tube. Twenty
microliters of 75% methanol was then added into the
tube and shaken briefly to mix. Each sample was then
sonicated in an ultrasonic cleaner (Delta DC400H) at a

frequency of 40 kHz at 25°C for 30 min. The extract was
centrifuged for 10 min at 3000 rpm and the supernatant
was then transferred into a 50 mL volumetric flask. The
procedure was repeated for one more time and the
supernatants were combined. The final volume was made
up to 50 mL with 75% methanol. The final combined
extract was filtered through a 0.45 |im PVDF syringer
filter (VWR Scientific, Seattle, WA) before analysis. An
aliquot of 10 \iL solution of each sample was used for
HPLC and HPLC-ESI-MS analyses.

Standards solutions

Stock standard solutions of the three accurately weighed
reference compounds were prepared in 75% methanol.
A standard mixture was obtained by mixing the individual
stock standard solution to give THSG at a concentration
of 252.5 mg/L, emodin at 138.325 mg/L and physcion at
15.4 mg/L. The standard mixture was diluted with 75%
methanol to appropriate concentrations for calibration
curves. The solutions were brought to room temperature
and filtered through 0.45 \im PVDF syringer filter and
an aliquot of 10 \iL of each solution was used for
HPLC analysis.

HPLC analysis

HPLC analyses were performed on a Waters 2695 HPLC
system equipped with Waters 2998 photodiode array
detector (PDA), Waters e2695 separations module and
column heater module. A Grace Alltima CI 8 column
(250 mm x 4.6 mm i.d., 5 \xm) was used. The mobile phase
consisted of 0.5% vlv formic acid aqueous solution (A)
and acetonitrile (B). The optimized elution conditions
were as follow: 0-22 min, 16% B; 22-45 min, 16-34% B;
45-60 min, 34-38% B; 60-70 min, 38-95%; 70-80 min,
95% B. The flow rate was 1 mL/min and the injection
volume was 10 [iL. UV spectra were acquired from 190 nm
to 400 nm. The autosampler and column compartment
were maintained at 25°C and 35°C, respectively.

HPLC-ESI-MS analysis

HPLC-ESI-MS analyses were performed on a TSQ
Quantum Access Max Triple Stage Quadrupole Mass
Spectrometer (Thermo Fisher Scientific Inc., Waltham,
MA, USA) with an Accela 1250 UHPLC system
equipped with an Accela 1250 photo diode array (PDA)
detector, an Accela HTC PAL autosampler, and an
Accela 1250 binary pump. The column and elution
conditions used were the same as those used in "HPLC
analysis" except that the flow rate was set at 0.25 mL/min
with a split ratio. Ultrahigh pure helium (He) and high
purity nitrogen (N 2 ) were used as collision gas and for
nebulizer, respectively. The optimized parameters in
negative/positive ion modes were as follows: ion spray
voltage, -2.5 kV/3.0 kV; auxiliary gas, 40 arbitrary

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Table 1 Collected information of the nineteen PMC samples and the content (%) of THSG, emodin and physcion in the

samples

Sample

Content (mg/g dry weight) (Mean ±

SD)

Origin

Type

Diameter

Moisture

No.

THSG

Emodin

Physcion

(mm)

content (%)

PMC-01

19.547 + 0.0016

0.791

± 0.0003

0.785 ±0.0001

Zhejiang

Raw Material a

3-9

11.10

PMC-02

23.766 ±0.01 16

1.295

± 0.0007

1 .394 ± 0.0005

Yunnan

Raw Material

3-9

10.18

PMC-03

0.742 ± 0.0007

0.023

± 0.0001

ND

Guangxi

Medicinal Slices b

7-12

8.23

PMC-04

1 .275 ± 0.0007

0.054

± 0.0001

0.043 ± 0.0000

Sichuan

Raw Material

0.5-2

9.32

PMC-05

0.351 ±0.0004

0.015

± 0.0000

ND

Sichuan

Raw Material

3-9

9.98

PMC-06

0.268 ±0.0001

0.074

± 0.0000

0.086 ± 0.0000

Jiangsu

Medicinal Slices

3-11

11.66

PMC-07

5.955 ± 0.0022

0.452

± 0.0002

0.592 ± 0.0005

Sichuan

Raw Material

4-8

10.09

PMC-08

11. 184 ±0.0070

0.424

± 0.0004

0.836 ± 0.0008

Hu'nan

Raw Material

2-5

8.06

L-PMC-01

1.195 ±0.0032

0.031

± 0.0000

0.1 02 ±0.0002

Guangxi

Medicinal Slices

11-26

7.66

L-PMC-02

0.427 ± 0.0003

0.010

± 0.0000

ND

Guangdong

Medicinal Slices

9-27

8.03

L-PMC-03

0.501 ±0.0002

0.025

± 0.0001

0.01 5 ±0.0001

Yunnan

Medicinal Slices

10-26

8.20

L-PMC-04

1.482 ±0.0011

0.039

± 0.0000

0.042 ± 0.0000

Guangxi

Raw Material

4-14

7.94

L-PMC-05

0.880 ± 0.0005

0.027

± 0.0000

0.01 3 ±0.0000

He'nan

Medicinal Slices

10-28

7.64

L-PMC-06

1.683 ±0.0014

0.014

± 0.0000

ND

Unknown

Medicinal Slices

12-26

9.25

L-PMC-07

ND

ND

ND

Guangxi

Raw Material

7-36

8.67

L-PMC-08

0.840 ± 0.0005

ND

ND

Hu'nan

Medicinal Slices

4-14

8.87

L-PMC-09

ND

ND

ND

Guangxi

Raw Material

8-23

8.53

L-PMC-10

ND

ND

ND

Unknown

Raw Material

7-15

7.62

L-PMC-1 1

41.361 ±0.0009

0.864

± 0.0009

1 .054 ± 0.0006

Unknown

Raw Material

2-6

8.74

a Tied into a bundle. Cut into small pieces and dried in shade places before experiments.
b Purchased from markets or pharmacies.

units; sheath gas, 15 arbitrary units; capillary temperature,
350°C; vaporizer temperature, 350°C; capillary offset, -35
V/18 V; tube lens offset, -33 V/102 V. Spectra were
recorded in the range of mlz 100-1000 for full scan data,
meanwhile, the normalized collision energy was set at 45%
for MS 2 data with dependant scan.

Quantitative analytical method validation

The limits of detection (LOD) and quantitation (LOQ)
were defined as the lowest concentrations of analytes in the
sample that can be detected and quantified, which were
determined on the basis of signal-to-noise ratios (S/N) at
3:1 and 10:1, respectively. Intra- and inter-day variations
were chosen to evaluate the precision of the developed
method. The intra-day variation was determined by
analyzing one of the mixed stock solutions (THSG at
50.5 mg/L, emodin at 27.665 mg/L and physcion at
3.08 mg/L) five times within one day. While for inter-day
variability test, the same solution was examined in
triplicate for three consecutive days. Repeatability was
confirmed with five different working solutions prepared
from sample PMC-01. Stability was tested with the same
sample solution at 0, 2, 4, 8, 12, 24 h.

Fingerprinting and principal component analyses

The data obtained from chromatographic fingerprints
were analyzed with Solo (Eigenvector Research, Inc.,
Wenatchee, WA) for chemometric analysis. Normalize
(2-Norm, length = 1) and mean center were used for
data reprocessing before principal component analysis
(PCA) was performed.

Results and discussion

Optimization of extraction method

The extraction solvents were optimized based on the
extraction efficiency of THSG. Four solvents, ethanol,
50% methanol, 75% methanol and methanol were
investigated with sonication at room temperature for
30 min. As a result, 75% methanol was proved to be
superior to other solvents (Additional file 1: Figure SI A
and Figure SI B), which was selected as the extraction
solvent. The optimal extraction times for 75% methanol
was further investigated. The powder of Polygoni
Multiflori Caulis (0.5 g) was extracted with 20 mL of 75%
methanol for three times (30 min for each time). It
showed that most THSG was extracted (> 99%) after the
second extraction. (Additional file 1: Figure SI C). Finally,

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the optimal extraction method was finalized, as described
in "Sample preparation".

Optimization of chromatographic conditions

To develop a reliable chromatographic fingerprinting
method, an optimized strategy for HPLC conditions was
performed. To obtain sharp and symmetrical peaks,
different mobile phase systems, including methanol-water,
acetonitrile-water, acetonitrile-water (containing 0.5%
formic acid, v/v) and acetonitrile-water (containing 0.1%
phosphoric acid, v/v) were tested. As a result, high
resolution, good baseline, sharp and symmetrical peaks
were obtained by using acetonitrile-water (containing 0.5%
formic acid, v/v) system. A few columns (Waters XBridge
Shield RP18, Waters XTerra RP18, Thermo Ascentis C18)
were screened before Grace Alltima CI 8 column
(250 mm x 4.6 mm i.d., 5 (im) was finally selected as
the column of choice. To obtain a sufficient large
number of detectable peaks on the chromatographic
fingerprints, PDA full scan (190-400 nm) was used
for investigating all the main peaks and finally 290 nm was
selected as detection wavelength. Representative chromato-
graphic fingerprint obtained from PMC-01 is shown in
Figure 1. Characteristic chemical compounds are marked
as 1 to 7. In quantitative analysis, THSG was monitored at
320 nm, meanwhile, emodin and physcion were monitored
at 290 nm. Different column temperatures at 20, 25, 30 and
35°C were also investigated. Although chromatograms
detected at different temperatures didn't show obvious
differences, 35°C was selected as the preferable one in order
to minimize the influences from room temperature
on the chromatograms. In the process of gradient
optimization, gradient time, gradient procedure and
initial composition of the mobile phase were taken
into consideration. Finally, the gradient procedure was
finalized, as described in "HPLC Analysis".

Assignments of the seven characteristic peaks

Figure 1 shows the seven characteristic peaks detected at
290 nm in PMC-01. The structural identification of each
peak was carried out by careful studies on MS and MS 2
spectra and by comparison with reference compounds and
literatures (Table 2). Under the optimized MS conditions,
both negative and positive ESI modes were used in
our experiment.

Peak 1 occurs at retention time of 19.6 min with maximal
UV absorption at 319 nm. In negative ion mode, the
deprotonated molecular ion at mlz 405 [M-H]~, formic acid
adduct ion at mlz 451 [M-H + HCOOH]" and 811
[2 M-H]~ were found in its MS spectrum. Fragmentation
of the ion at mlz 405 [M-H]~ yielded a product ion at mlz
243 arising from the loss of a glucosyl (-C 6 H 10 O 5 ) unit. In
positive ion mode, the protonated molecular ion at mlz
407 [M + H] + and a sodium adduct ion at mlz 429
[M + Na] + were found in its MS spectrum. The MS 2
fragmentation of the ion at mlz 407 was further investi-
gated and a dominant product ion at mlz 245 [M + H-
glucosyl] + was observed, corresponding to the loss of the
glucosyl unit (162 amu). This peak was unequivocally
identified as THSG by comparison with MS data of the
standard as well as literatures [11-14].

Peak 2 shows the retention time of 45.1 min with
maximal UV absorption at 281 nm. This peak gave
[M-H]~ ion at mlz 431 and [2 M-H]~ ion at mlz 863
in MS spectrum in negative ion mode (Figure 2A).
The ion at mlz 431 generated a series of fragment
ions in its MS 2 spectrum at mlz 269 [M-H-glucosyl]",
241 [M-H-glucosyl-CO]-, 225 [M-H-glucosyl-C0 2 ]~,
197 [M-H-glucosyl-C0 2 -CO]- and 182 [M-H-glucosyl-
C0 2 -CO-CH 3 ]" (Figure 2B). In positive ion mode,
peak 2 produced a very week [M + H] + ion but yielded
prominent ions at mlz 455 [M + Na] + , 887 [2 M + Na] + and
271 [M + H-glucosyl] + in its MS spectrum (Figure 2C).

0.24
0.22
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02

5

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

Minutes

0.00 65.00 70.00 75.00 80.00

Figure 1 Representative chromatographic fingerprint obtained from PMC-01 detected at 290 nm. Seven characteristic peaks are marked
as 1 to 7.

Table 2 Assignments of the seven characteristic peaks by HPLC/UV/MS

No.

RT

(min)

UVA max
(nm)

MS in Neg. mode

MS 2 in Neg. mode

MS in Pos. mode

MS 2 in Pos. mode

Assignment

References

1

19.6

319

405 [M-H]"

451 [M-H + HCOOH]"

811 [2 M-H]"

243 [M-H-glucosyl]"

407 [M + H] +

429 [M + Naf

245 [M + H-glucosylf

245 [M + H-glucosylf

THSG

[11-14]

2

45.1

281

431 [M-H]"
863 [2 M-H]"

182 [M-H-glucosyl-C0 2 -CO-CH 3 ]"
197 [M-H-glucosyl-C0 2 -CO]"
225 [M-H-glucosyl-C0 2 ]"
241 [M-H-glucosyl-CO]"
269 [M-H-glucosyl]"

433 [M + H] +
455 [M + Naf
887 [2 M + Naf
271 [M + H-glucosylf

Emodin-8

-0-,6-D-glucoside

[11,13-16]

3

48.2

281

517 [M-H]"
473 [M-H-C0 2 ]"

225 [M-H-malonylglucosyl-C0 2 ]"
269 [M-H-malonylglucosyl]"

541 [M + Naf

271 [M + H-malonylglucosylf

Emodin-8

-0-(6'-0-malonyl)-/3-D-glucoside

[14]

4

50.5

270

445 [M-H]"

491 [M-H + HCOOH]"
283 [M-H-glucosyl]"

212 [M-H-glucosyl-CH 3 -2CO]"
240 [M-H-glucosyl-CH 3 -CO]"
253 [M-H-glucosyl-2CH 3 ]"
268 [M-H-glucosyl-CH 3 ]"
283 [M-H-glucosyl]"

469 [M + Naf

285 [M + H-glucosylf

Physcion-

8-0-/3-D-glucoside

[11,13-16]

5

55.8

270

487 [M-H]"

533 [M-H + HCOOH]"

283 [M-H-acetylglucosyl]"

240 [M-H-acetylglucosyl-CO-CH 3 ]"

511 [M + Naf

285 [M + H-acetylglucosylf

Physcion-

8-0-(6'-0-acetyl)-/3-D-glucoside

[11,16]

6

70.1

222, 288

269 [M-H]"

182 [M-H-CO-C0 2 -CH 3 ]"
225 [M-H-C0 2 ]"
241 [M-H-CO]"

271 [M + Hf

Emodin

[11-17]

7

73.8

223, 286

283 [M-H]"

212 [M-H-2CO-CH 3 ]"
240 [M-H-CO-CH 3 ]"
255 [M-H-CO]"

285 [M + Hf
307 [M + Naf

Physcion

[12-17]

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[M-H]-

A

500000 :
450000 :
400000 :
350000 :
300000 :
250000 :
200000 :

[2M-H]"

Ij499.67 560.46 f

3 617.90 642.58

450 500

F:\TW_201 301 02\...\Neg\N„SWT_0306_2
N_SWT_0
36000-1

2013-3-6 16:17:07

[M-H-Glucosyl]

B

431

28000-
26000-
24000-
22000-
20000-

-CO

-co 2

6000-
4000-
2000-

-CH 3 19712

i6.87 162.97 170.68 174.20

200 210

-CO

J3.65 ] 221.3!

250.31 253.19 262.8'

230 240

900000 :
850000 :
800000 :
750000:
700000:
650000:
600000:
550000 :
500000-
450000 :
400000-
350000 :
300000:
250000:
200000:
150000:

mJ^M

8 [M+H-Glucosyl] +

[M+Na] +

31 1 .70
297.37

m \ mW

i33 58 370.77 '

,2.18 527 ^r 59365

I I, 552.75 I 615.04

[2M+Na] +

150 200 250

400 450 500 550

700 750

900 950

Figure 2 Mass spectra of emodin-8-O-jS-D-glucoside (peak 2) in (-)-ESI-MS (A), (-)-ESI-MS 2 (B) and (+)-ESI-MS (C).

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Consequently, it was characterized as emodin-8-0-/?-
D-glucoside, of which some of the MS fragmentation
behaviors were described in published papers [11,13-16].

Peak 3 occurs at retention time of 48.2 min with
maximal UV absorption at 281 nm. Ions at mlz 517 and
473 were observed in its MS spectrum in negative ion
mode, which were speculated as [M-H]" and [M-H-C0 2 ]~
ions, respectively. The ions at mlz 269 [M-H-malonyl-
glucosyl]" and 225 [M-H-malonylglucosyl-C0 2 r were
found in its MS 2 spectrum. Protonated molecular ion was
not found in its MS spectrum in positive mode, but
sodium adduct ion at mlz 541 [M + Na] + and the one
which lost a malonylglucosyl unit at mlz 271 [M + H-
malonylglucosyl] + were predominant. This peak was
tentatively identified as emodin-8-0-(6'-0-malonyl)-/?-D-
glucoside based on its MS data and the literature [14].

Peak 4 occurs at retention time of 50.5 min with
maximal UV absorption at 270 nm. In negative ion
ESI experiments, it yielded prominent deprotonated
molecular ion at mlz 445 [M-H]", formic acid adduct
ion at mlz 491 [M-H + HCOOH]" and the ion at mlz 283
[M-H-glucosyl]". The MS 2 spectrum of the ion at mlz 445
showed characteristic ions at mlz 283 [M-H-glucosyl]",
268 [M-H-glucosyl-CH 3 ]", 253 [M-H-glucosyl-2CH 3 ]",
240 [M-H-glucosyl-CH 3 -CO]" and 212 [M-H-glucosyl-
CH 3 -2CO]". In positive ion mode, the protonated
molecular ion at mlz 447 was not found but the sodium
adduct ion at mlz 469 [M + Na] + and the ion at mlz
285 [M + H-glucosyl] + were observed as predominant
ions in MS spectrum. This peak was tentatively identified as
physcion-8-0-/?-D-glucoside based on the data mentioned
above and the literatures [11,13-16]. A proposed fragmenta-
tion pathway for the deprotonated ion at mlz 445 [M-H]" of
physcion-8-0-/?-D-glucoside is shown in Figure 3.

Peak 5 shows the retention time of 55.8 min with
maximal UV absorption at 270 nm. Characteristic ions

at mlz 487 [M-H]", 533 [M-H + HCOOH]" and 283
[M-H-acetylglucosyl]" were produced from this peak in
MS spectrum in negative ion mode. The deprotonated ion
at mlz 487 [M-H]" gave a predominant ion at mlz 240 in
MS 2 spectrum resulting from the losses of a acetylglucosyl
unit, a neutral molecular of CO and a methyl group.
In positive ion mode, we did not find the protonated
molecular ion, however it yielded a predominant sodium
adduct ion at mlz 511 [M + Na] + and the ion at mlz 285
by losing a acetylglucosyl unit. By comparison with the
reported paper [11,16], the peak was identified as
physcion-8-0-(6 ' -0-acetyl)-/?-D -glucoside.

Peak 6 was eluted at retention time of 70.1 min
with maximal UV absorption at 222 and 288 nm,
which produced the [M-H]" ion at 269 in the MS
spectrum in negative ion mode. It further gave fragment
ions at mlz 241 [M-H-CO]", 225 [M-H-C0 2 ]" and 182
[M-H-CO-C0 2 -CH 3 ]" in the MS 2 spectrum. In positive
ion mode, the peak yielded weak protonated molecular
ion at mlz 271 [M + H] + in MS spectrum, and no
useful information was obtained in its MS 2 spectrum.
By comparison with MS behaviors of the standard
and the literatures [11-17], the peak was unequivocally
identified as emodin.

Peak 7 was eluted at retention time of 73.8 min with
maximal UV absorption at 223 and 286 nm. Deprotonated
molecular ion at mlz 283 [M-H]" was observed in its MS
spectrum in the negative ion mode, which further
generated a predominant ion at mlz 255 in MS 2
spectrum owing to the loss of a neutral CO molecule.
Other fragment ions at mlz 240 and 212 were also
observed owing to the successive losses of a methyl
unit and a CO molecule from 255. In positive ion
mode, protonated molecular ion at mlz 285 [M + H] + and
sodium adduct ion at mlz 307 [M + Na] + were observed
in MS spectrum of the peak. Based on the MS data

OH O OH

-CH,

-co

Figure 3 The proposed fragmentation pathway for the deprotonated ion at m/z 445 [M-H]" of physcion-8-O-jS-D-glucoside.

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reported in publications [12-17] and the comparison with
the standard, it was unequivocally identified as physcion.

Method validation

Calibration curves, LODs and LOQs

The calibration curve of each compound was performed
with six appropriate concentrations in duplicate and
constructed by plotting the peak areas versus the
concentrations. As shown in Table 3, all calibration
curves showed good linear regression (I? 2 > 0.9990) in a
relatively wide range. The stock solution of each reference
compound was further diluted to a series of concentrations
with 75% methanol for LOD and LOQ. LODs for THSG,
emodin and physcion were 98.08 ng/mL, 44.26 ng/mL and
56.77 ng/mL, respectively, and LOQs for THSG, emodin
and physcion were 404.03 ng/mL, 180.56 ng/mL and
132.20 ng/mL, respectively, with injection volume of 10 //L.

Precision, repeatability and stability

The RSDs of the retention times and peak areas of
the three analytes were taken as the measurements of
precision and stability. The RSDs of the retention
times and contents of the three analytes in dried
samples (mg/g) were taken as the measurements of
repeatability. As shown in Table 4, the overall RSD
values were less than 3.00%, indicating that the developed
method was satisfactory on quantification of THSG,
emodin and physcion in PMC samples.

Quantification of THSG, emodin and physcion in PMC
samples

The established HPLC-UV quantitative analytical method
was successfully applied for simultaneous quantification
on the three compounds in nineteen PMC samples
(eight were from mainland China, and eleven were
from local pharmacies of Hong Kong). The contents
(mg/g dry weight) were calculated and summarized
(n = 2) in Table 1.

Firstly, the results showed that the contents of each
compound in different PMC samples varied markedly. To
our surprise, the contents of THSG, emodin and physcion
ranged from 0.268 to 41.361 mg/g, 0.010 to 1.295 mg/g

and 0.013 to 1.394 mg/g, respectively. In addition, THSG
was not detected (below LOD) in L-PMC-07, L-PMC-09
and L-PMC-10, emodin was not detected in L-PMC-07,
L-PMC-08, L-PMC-09 and L-PMC-10, and physcion was
not found in PMC-03, PMC-05, L-PMC-02, L-PMC-06,
L-PMC-07, L-PMC-08, L-PMC-09 and L-PMC-10.
The results indicated that significant differences of
the concentrations of each compound in different PMC
samples were found.

Secondly, according to the regulation of China
pharmacopoeia (2010 edition) that the content of THSG
in dried PMC sample should not be less than 0.20%
(2.0 mg/g), only five samples in our study, including
PMC-01, PMC-02, PMC-07, PMC-08 and L-PMC-11,
were definitely qualified raw medicinal materials for
clinic use. It was worth mentioning that one of the
eleven local PMC samples from Hong Kong, L-PMC-11,
had the highest content of THSG in all the tested
samples, which was also the only one qualified sample
from local pharmacy of Hong Kong. Emodin and
physcion are not the specified chemical markers in
China pharmacopoeia, but they usually exist in the plants
from family Polygonaceae, which were also quantified in
PMC or its related commercial product [18,19]. The data
obtained in the present study showed that except the
samples in which emodin were not detected, the content
of emodin was the highest in PMC-02, however, the
lowest, in L-PMC-02. In the same way, the content of
physcion in PMC-02 was the highest and the one in
L-PMC-05 was the lowest. The results also indicated
that emodin and physcion were not the dominant
chemical compounds in PMC compared with THSG.

Thirdly, the PMC samples tested in the present study
were mainly from southlands of China. L-PMC-11 was
found to have the highest content of THSG at 41.361 mg/g
and relative higher contents of emodin (0.864 mg/g) and
physcion (1.054 mg/g) in all the tested samples. But
its origin was unknown. The sample from Yunnan,
numbered PMC-02, had the highest contents of emodin
(1.295 mg/g) and physcion (1.394 mg/g) as well as the
second highest content of THSG (23.766 mg/g) in all the
samples. However, the samples, in which the three analytes

Table 3 Regression data, LODs and LOQs for the three analytes tested in HPLC-UV chromatograms

Analyte

Calibration curve 3

R 2

Linear range (mg/L)

LOD b (ng/mL)

LOQ c (ng/mL)

1

y= 29885 x- 210960

0.9990

0.404-252.5

98.08

404.03

2

y = 38332 x + 23549

0.9999

0.0443-138.325

44.26

180.56

3

y= 22565 x- 1352.2

0.9997

0.123-15.4

56.77

132.20

1:THSG.
2: emodin.
3: physcion.

a y is the peak area in UV chromatograms, x is the concentration (mg/L) of the analyte.

b LOD refers to the limit of detection, s/n = 3.

c LOQ refers to the limit of quantification, s/n = 10.

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Table 4 Results of precision, repeatability and stability of the three analytes, expressed as RSD (%)

Analyte Precision Repeatability (n = 5) Stability (n = 6)

Intra-day RSD (%) (n = 5) Inter-day RSD (%) (n = 9) RSD (%) RSD (%)

t R a PA b t R PA t R Contents (%) t R PA

1 0.58 1.24 2.29 1.98 0.78 1.25 1.69 2.85

2 0.62 0.88 1.98 2.01 0.51 1.69 0.96 2.31

3 0.49 1.58 1.86 2.89 0.66 2.08 1.33 2.44

a Refers to retention time.
b Refers to peak area.

were not detected, were from different origins. Seeing from
the results, we find that the qualities of PMC samples col-
lected for the present study do not have necessary relations
with their origins.

The last but the most important, PMC is the dried caulis
of Polygonum multiflorum Thunb., which is a perennial
plant from family Polygonaceae. One of the descriptions of
PMC in China pharmacopeia (2010 edition) is that their
diameters are between 4 and 7 mm, but the diameters of
PMC on market have great differences. The information
about diameters of PMC samples in our study are summa-
rized in Table 1. The contents results indicated that the

diameters of the five qualified PMC samples, including
PMC-01, PMC-02, PMC-07, PMC-08 and L-PMC-11,
basically fell in the defined range (each sample had a
few stems of which the diameters were out of the
range) (Figure 4A). They also had higher contents of
emodin and physcion than others. The diameters of
L-PMC-07, L-PMC-09 and L-PMC-10, in which all
the three analytes were undetectable, all exceeded the
defined range. To our surprise, no obvious peak was
detected in these samples (Figure 4B). As for PMC-05,
PMC-06, L-PMC-04 and L-PMC-08, just a few branches of
each sample were thin but most of them were thick,

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Eigenvalues for PCM Data.xlsx

S

u

3 4 5

Principal Component Number

Figure 5 The cumulative variance of the seven generated
principal components. The first two components account for
94.75% of total variance.

resulting in their unqualified qualities. Neither the sample
(PMC-04) with diameter value lower than 4 mm nor the
samples with diameter values more than 7 mm (PMC-03,
L-PMC-01, L-PMC-02, L-PMC-03, L-PMC-05 and L-PMC
-06) were unqualified. But it was worth noting that PMC-04
with the lowest diameter value had relative higher content
of THSG in all the unqualified samples. So, we speculated
that the content of THSG would increase with its growth.
When the diameter values of its stems reached the range
between 4 and 7 mm, its content of THSG would met with
CP s requirement and then it was the best harvesting time.
In addition, as shown in Table 1, eight out of nineteen in
our tested PMC samples were medicinal slices. All of them
are unqualified ones according to the specification in CP
(2010 edition), but we don't think their qualities have
positive correlations with processing methods since raw

materials of PMC are just cut and dried in shade
places to get medicinal slices generally. We speculate
that it is still the diameters of stems' of the raw materials
that influence their qualities.

Fingerprinting and principal component analyses

Although the quantification results can confirm the
contents of THSG, emodin and physcion in a PMC
sample, there is no way to know intuitively how similar a
PMC sample to another one on the whole just by quantifi-
cation a few compounds. Fingerprinting and chemometrics
analyses, however, can show the chemical similarities be-
tween one and another one holistically and visually. Princi-
pal component analysis, one of the chemometrics, is an
unsupervised mathematical procedure that transforms a
number of possibly correlated variables into a smaller num-
ber of uncorrelated variables called principal components.
Its operation can be thought of as revealing the internal
structure of the data in a way which best explains the
variance.

Seven peaks marked 1 to 7 were selected as characteristic
peaks in chromatographic fingerprints in the present study
(Figure 1). Although peak 2 ~ peak 5 were tentative identi-
fied as emodin-8-0-/?-D-glucoside, emodin-8-0-(6'-0-
malonyl)-/?-D-glucoside, physcion-8-0-/?-D-glucoside and
physcion-8-O- (6' -acetyl) -/?-D-glucoside respectively based
on the data in Table 2 and published literatures, due to the
unavailability of reference compounds, they were not quan-
tified. So, the variables of each sample consisted of contents
of peak 1, peak 6 and peak 7 and PA/W (peak area divided
by sample weight) values of peak 2 ~ peak 5. The data were
exported to Excel (Microsoft, Inc., Belleview, WA) to form
a two-dimensional matrix (nineteen samples versus seven
variables) which was then exported to SOLO for PCA. A
two-component (the first two components) model cumula-
tively accounted for 94.75% of total variance (Figure 5),

r4 0
U

3 -1

Samples/Scores Plot of PCM Data.xlsx

! •PMC-07-^

s' 1 «>MC-01

/ 1 \
/ - \ -

.ftffefe - PMC - 08

L l _

\ «P

\

\

\

\

\

\ ^

7

/

/

/

/

•PMC-02

-2 0 2

Scores on PC 1 (82.63%)

Figure 6 PCA scores plot of PMC samples.

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based on which PCA scores plot (Figure 6) was
generated. From the scores plot, we can see intuitively a
very interesting phenomenon that PMC-01, PMC-02,
PMC-07, PMC-08 and L-PMC-11, which were regarded
as the only five qualified samples in the present study, are
separated very well with other samples in PCI. They all
get PCI scores above zero, however, others obtain PCI
scores below zero. What's more, L-PMC-11 is located
outside the ellipse (95% confidence interval) because
of its the highest content of THSG.

To find out variables contributing to the significant
differences between different PMC samples, PCI and
PC2 loadings plots were generated. PCI loadings plot
(Figure 7A) indicates peak 2, peak 4, peak 6 and peak 7
are mainly responsible for the separation of samples on
PCI (P < 0.05). What it means is that higher PA/W

values of peak 2 and peak 4 and higher contents of peak
6 and peak 7 will give higher PCI scores, moving the po-
sitions of the samples to the right on PCA scores plot.
PMC-01 gets the highest PA/W value of peak 2 in all
the samples. PMC-02 gets the highest PA/W value of
peak 4 and the highest contents of peak 6 and peak 7 in all
the samples. So, the two samples are placed in the right-
most positions in scores plot. In the same way, L-PMC-11,
PMC-07 and PMC-08 have higher contents of peak 6
and peak 7 or higher PA/W values of peak 2 and
peak 4, making them get higher PCI scores than others
except PMC-01 and PMC-02.

According to PC2 loadings plot, peak 3 and peak 5
mainly contribute to high PC2 scores, however, peak 1,
peak 6 and peak 7 contribute to low PC2 scores (P < 0.05).
PMC-07 has the highest PA/W value of peak 3 and the

Variables/Loadings Plot for PCM Data.xlsx

-0.8

Variables/Loadings Plot for PCM Data.xlsx

4 5
Variable

Figure 7 PCI (A) and PC2 (B) loadings plots of the seven variables.

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second highest PA/W value of peak 5. PMC-01 has
the highest PA/W value of peak 5 and the second
highest PA/W value of peak 3. So, they are positioned on
the top in scores plot, having higher PC2 scores. The
highest content of peak 1 was found in L-PMC-11,
and then in PMC-02. The highest PA/W value of
peak 6 and the highest content of peak 7 were both
obtained in PMC-02, and then in L-PMC-1L The
above two reasons lead to the positions of the two
samples at the bottom.

Other samples are clustered tightly around the corner
due to their similar and low contents or PA/W values of
the seven peaks.

All in all, scores plot shows the distributions of
the tested samples intuitively and clearly, meanwhile,
loadings plots indicate the influences of the variables
on the positions of PMC samples. From the scores
obtained in the present study, qualified and unquali-
fied PMC samples can be distinguished easily and
efficiently.

Conclusions

For the first time, systematic HPLC/UV/MS chromato-
graphic fingerprinting and quantitative analytical methods
combined with principal component analysis were devel-
oped to analyze different PMC samples. The contents of
THSG were found to have surprising relevance with the
samples' diameters. Diameters of the five qualified PMC
samples basically fell in the specified range, which also had
higher contents of emodin and physcion than others.
However, diameters of the unqualified PMC samples
generally exceeded the specified range. Seven characteristic
peaks in chromatographic fingerprints marked 1 to 7 were
identified, and based on the contents or PA/W values of
the seven variables, PCA scores plot was generated. The
finding in the present study provides a scientific basis for
collecting PMC in the best time, and with the aid of PCA,
unqualified PMC samples can be singled out from qualified
ones easily and efficiently.

Additional file

Additional file 1: Figure SI A. Chromatograms of Polygoni Multiflori
Caulis extracted with different solvents. Figure S1 B. The peak areas of
THSG in different chromatograms of Polygoni Multiflori Caulis extracted
with different solvents. Figure S1 C. The chromatograms of Polygoni
Multiflori Caulis extracted with 75% methanol for three times.

Competing interests

The authors declare that they have no competing interests.
Authors' contributions

YSC and YLH initiated and design the study. The extraction and method
developments were conducted by YZ and CPK. YZ drafted the manuscript.
All authors contributed to data analyses and finalized the manuscript. All
authors have read and approved the final version.

Acknowledgement

We like to thank Taiwan Department of Health Clinical Trial and Research
Center of Excellence, China Medical University Hospital (DOH99-TD-B-1 11-
004) for providing UPLC/MS facility for this study. The authors also like to
acknowledge the grant support from Chinese Materia Medica Standard
Office, Department of Health, Hong Kong for this study.

Author details

department of Chinese Pharmaceutical Sciences and Chinese Medicine
Resources, College of Pharmacy, China Medical University, Taichung 40402,
Taiwan, department of Nursing, Hsin Sheng College of Medical Care and
Management, Taoyuan 32544, Taiwan. 3 Chinese Crude Drug Pharmacy, China
Medical University Hospital, Taichung 40402, Taiwan, department of
Nursing, Hungkuang University, Taichung 43302, Taiwan.

Received: 17 April 2013 Accepted: 19 June 2013
Published: 24 June 2013

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doi:1 0.1 186/1 752-1 53X-7-1 06

Cite this article as: Zhao et al.: Quality assessment on Polygoni Multiflori
Caulis using HPLC/UV/MS combined with principle component analysis.

Chemistry Central Journal 201 3 7:1 06.

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